Paediatrics (RCH) - Theses
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ItemCharacterisation of immune differences in the development of food allergy in infants( 2013)Food allergy often develops early in life and has a major impact on the quality of life of the infant. It is characterised by a dysregulation of the immune response in which there is increased production of Th2 cytokines, resulting in B cell class-switching and production of food specific-IgE antibodies. Sensitisation to food allergens indicates the production of food specific IgE (sIgE) antibodies, but is not a definite indicator of a clinically allergic reaction upon ingestion. Although some individuals who produce food specific-IgE antibodies will react on consumption of the food in question (food allergic), others are sensitised but can consume the food without any adverse consequences (food sensitised). The mechanisms underpinning why some food-sensitised children are tolerant and some are allergic have not been fully elucidated, and it is postulated that tolerance to food antigens arises from regulatory T cells (Tregs) induced in the periphery, and that a diminished capacity to produce these cells results in the development of food allergy. Furthermore, due to the rapid rise in rates of food allergy combined with the lag in training new allergists, allergy services are currently overwhelmed, with patients having limited access to oral food challenge (OFC) tests. Although OFC remain the gold standard for diagnosing food allergy, these are time consuming, costly, and associated with a risk of anaphylaxis. While 95% positive predictive values thresholds for sIgE can assist with identifying increased likelihood of allergy among those who are sensitised, there are no specific biological markers that differentiate between allergic and sensitised individuals. Therefore, improved correlates of allergy status are required. This thesis aimed to investigate the key immunological differences that distinguish between the clinical phenotypes of food sensitisation and food allergy in one-year old infants, using samples from the HealthNuts study (a population-based cohort study of 5,276 infants recruited at 12 month old infants). In particular the role of Treg cells in the breakdown of tolerance in food allergics was studied. We explored whether there were any immune differences in the blood which could be utilised as biomarkers to distinguish allergic subjects from sensitised tolerant subjects. We determined that food allergic infants have a reduced capacity to regenerate their pool of Foxp3+ Treg cells following in vivo allergen stimulation in the form of an OFC compared to food sensitised tolerant infants. We also found a reduced level of circulating plasma IL-10 to further support the notion that the development of food allergy is the result from the failure to develop oral tolerance to the food antigen. Furthermore, we established that there were immunological differences between egg and peanut allergy, including different Treg and plasma cytokine profiles. Infants with egg allergy had significantly higher plasma levels of both IL-13 and IL-12, and lower levels of IL-10 compared to infants with peanut allergy. Unlike the peanut allergic infants, the kinetics of activated Tregs from egg allergics remained unchanged throughout the course of the 6 days in culture following allergen exposure. Together with the plasma cytokine profiles, this suggests that the development of egg allergy may be the result of a ‘temporary’ immunological response. The symptoms of egg allergy are generally not as severe as peanut allergy, which has the highest rate of food related anaphylaxis. Therefore, suggesting that the immune mechanisms involved may be dependent on the type of food allergy. In terms of using plasma cytokines biological markers that differentiate between allergic and sensitised individuals, we found that key Th1 and Th2 cytokines were unable to separate the clinical phenotypes of food allergy. In order for the plasma cytokine work to be useful as a tool for diagnosing food allergy, more work is needed to develop and establish relevant cytokine profiles for the various characteristics of food allergy. An example of developing a model by combining other immunological data points was demonstrated in chapter 3, using both peanut component sIgE (Ara h2) and whole peanut sIgE along with peanut SPT results to determine an improved algorithm of diagnosing peanut allergy which would reduce the number of current peanut OFCs by up to 3-4 fold. In summary, the results described in this thesis outline several immunological differences between the three clinical phenotypes of food allergy examined, and further work into understanding the mechanisms will help differentiate these phenotypes for i) diagnostic purposes; and more importantly ii) treatment of allergic diseases.
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ItemRemote ischaemic preconditioning-evoked intracellular signalling pathways in cardiac maturation and disease( 2013)Background & Aims: Children with congenital heart defects are burdened with additional stress on the myocardium, which is exacerbated by cardiopulmonary bypass (CPB) and cardiac surgery. Ischaemic preconditioning (IPC) induced by brief, intermittent periods of ischaemia and reperfusion (IR) on the coronary vasculature can activate intrinsic protective mechanisms. However, remote IPC (RIPC) induced by inflating and deflating a standard blood-pressure cuff attached to a limb is a practical, non-invasive and clinically applicable model for protection against sustained ischaemic injury. RIPC regulates phosphorylation of key intracellular proteins that are recruited by IR and propagate signalling for metabolic control of the heart. Agonism of G-protein coupled receptors promotes protein signalling via key kinases such as Akt, p38MAPK, GSK3β, and HSP27 amongst other important effectors for cell survival against IR injury. However, most RIPC studies have focussed on adult myocardium. There is a dearth of such studies in immature myocardium. Thus, the aims of this Thesis were to: • test the efficacy of RIPC in a double-blind randomised trial in patients undergoing cardiac surgery for tetralogy of Fallot (ToF) and to measure key signalling protein kinases; • determine whether these signalling pathways are developed in murine neonatal hearts, and to compare their activation and ability to functionally recover after sublethal IR compared to adults; • determine the efficacy of RIPC in immature (4 weeks) and adult (12 weeks) hearts to functionally recover and express kinase signalling proteins after sublethal IR; • examine the effects of chronic hypoxia and fentanyl on immature cardiomyocytes on modulating kinase signalling proteins; • characterise the ability for advanced age (72 weeks) murine hearts to functionally recover and express kinase signalling proteins after sublethal IR. Methods: Limb RIPC was induced by four 5 min periods of inflation (ischaemia) and deflation (reperfusion) of a pressure cuff. IR occurred in ToF neonates during cardiac surgery utilising CPB, and in murine hearts during Langendorff-mode isolated heart perfusion. Resected right ventricular outflow tract myocardium from neonates and murine heart protein homogenates were assayed for protein expression by western immunoblotting. Effects of chronic hypoxia and fentanyl were studied in immature cardiomyocytes differentiated from murine P19 cells. Results: The major findings from this Thesis are: • remotely preconditioned ToF neonates did not have different expression of pro-survival signalling proteins relative to sham controls, which had a high proportion of phospho-kinase activation masking the effects of RIPC; • complex proteomic changes and a greater ability to functionally recover from sublethal IR were evident in immature murine hearts relative to the adult; • remotely preconditioned immature murine hearts had improved left ventricular (LV) functional recovery and an increase in the total available pool of protein kinases available for phosphorylation; • immature cardiomyocytes exposed to chronic hypoxia had an increase in total abundance of protein kinases available for phosphorylation; hypoxia blunted the effect of the opioid receptor agonist fentanyl; • advanced age hearts had impaired LV functional recovery, with a greater propensity for apoptosis and equitable levels of necrosis and autophagy, relative to adults. Conclusions: A protective role exists for RIPC in immature murine hearts exposed to IR despite undetectable protection in RIPC neonates undergoing ToF surgery. An extrapolation of these findings indicates confounders related to cyanotic disease progression (adaptation to chronic hypoxia) and pharmacological intervention may limit protection by RIPC in the surgical setting.